In short, we know almost nothing about the population structure, metabolic strategies, community composition, and global biogeochemical influence of the marine deep biosphere. We also know almost nothing about how the chemical and physical characteristics of subseafloor sediments control the microbial communities and activities that occur within those sediments. Consequently, we also know little about how modern microbial communities are constrained by past oceanographic history. If we are to develop a coherent understanding of the microbial communities that are deeply buried in marine sediments, a focused and interdisciplinary program of deep biosphere study is required. Leg 201 presents such a program.

Sampling of the Leg 201 sedimentary environments allows us to document the activity, composition, and biogeochemical effects of the subsurface biosphere in environments representative of essentially the entire range of subsurface conditions that can be found in relatively cool (2°25°C) marine sediments. These include equatorial Pacific sediments typical of the open ocean, Peru margin sediments typical of a nearshore upwelling regime, and Peru Basin sediments. Much of the geochemical and sedimentological character of these sediments has been documented during previous ODP and DSDP legs (DSDP Leg 34, Peru margin ODP Leg 112, and equatorial Pacific ODP Leg 138) (Yeats, Hart, et al., 1976; Mayer, Pisias, Janecek, et al., 1992, Suess, von Huene, et al., 1990; Pisias, Mayer, Janecek, et al., 1995). In short, several widely different marine sedimentary regimes were explored during this single drilling leg. Few regions in the world contain within a relatively short distance so many marine sedimentary regimes that have been so well characterized.

The environments that were examined include (1) carbonate and siliceous oozes of the equatorial Pacific, (2) clays and nannofossil-rich oozes of the Peru Basin, (3) organic-rich silty sediments of the shallow Peru shelf, and (4) a hydrate-rich deepwater sequence off the continental shelf of Peru (see Fig. F1).

The first two environments are characteristic of open-ocean sedimentary regimes. Leg 138 studies identified the presence of subsurface microbes throughout the sediment column in this equatorial Pacific region (Cragg and Kemp, 1995). Shipboard chemical analyses from Legs 138 and 34 (Pisias et al., 1995; Yeats, Hart, et al., 1976) suggest that the deeply buried microbial communities of these two regions rely primarily on sulfate and manganese reduction, respectively. Despite these studies, the subsurface extent of electron acceptors with similar or intermediate standard free-energy yields (nitrate, oxygen, and iron oxides) in these regions was unknown prior to Leg 201.

The second two environments are characteristic of ocean-margin regimes. Studies of Leg 112 samples identified abundant subsurface microbes in Peru shelf sediments (Cragg et al., 1990). At all sites but one, these shallow-water sediments and the deepwater hydrate-rich sediments are rich in dissolved sulfate at shallow burial depths (down to a few meters below seafloor) and rich in methane at greater burial depths (starting a few meters below seafloor or tens of meters below seafloor) (Suess, von Huene, et al., 1990). The remaining site is sulfate rich and methane poor throughout the targeted drilling interval, thus indicating relatively low microbial activity.

Subsurface flow affects the subsurface environment at both the shallow-water Peru shelf sediments and the open-ocean equatorial Pacific sites. In the former region, it is brine flow through the sediments. In the latter region, it is seawater flow through the underlying crust and perhaps the deepest sediments.

Scientific Objectives

The overarching objective of Leg 201 is to investigate the nature, extent, and biogeochemical consequences of microbial activity in several different deeply buried marine sedimentary environments.

During Leg 201, we addressed several fundamental questions about the deeply buried biosphere:

Are different sedimentary geochemical regimes characterized by completely different microbial communitiesor merely by shifts among the dominant species and different levels of community activity?

How does the transport of electron acceptors, electron donors, and, potentially, of microbes through deep sediments affect community structure and sediment chemistry?

To what extent do past oceanographic conditions affect microbial communities now active in deep-sea sediments?

How do biogeochemical processes of the deep subsurface biosphere affect the surface world?

Several aspects of these questions require extensive postcruise research to fully address. This reliance on postcruise research is necessary for at least two reasons. First, some experiments initiated during the cruise will still take months (radiotracer experiments) or years (cultivation experiments) to complete. Second, some key studies, such as genetic assays of the microbial communities and isotopic studies of biogeochemical fluxes, will be undertaken postcruise because they require technical facilities and expenditures of time beyond those available to a shipboard party during a single cruise.

Despite these limitations, other aspects of the above questions were successfully addressed during Leg 201. In particular, shipboard biogeochemical, geophysical, and sedimentological studies provide new understanding of the effects of pore water chemistry, sediment composition and structure, hydrologic flow, and past oceanographic conditions on metabolic diversity, microbial activities, and the nature of metabolic competition in these subsurface environments. These shipboard studies improve ourunderstanding of how deep subsurface biogeochemical processes affect both their local environments and the surface world.

Scientific Approaches

The study of deep subsurface microorganisms and their activity is a methodological and experimental challenge at the frontiers of modern life and earth sciences. Leg 201 is the first deep-sea drilling expedition to be primarily focused on subsurface microbial communities and their geochemical activities. Many of the studies carried out during this cruise were undertaken by ODP shipboard scientists for the first time. Many of these approaches had not been previously used to study the deep biosphere. A number of methods and concepts had to be further developed, refined, or even completely changed during the expedition. The scientific approaches were consequently chosen on the basis of extensive discussions and experiences of many colleagues and are still very much in the development phase. Some of these approaches may need further refinement before they are recommended for future routine application.

The research objectives of Leg 201 required shipboard scientists to address the following specific questions regarding the subseafloor sedimentary biosphere:

What are the physical-chemical conditions that support or limit microbial life at depth in marine sediments?

What are the microorganisms that inhabit these thousand-year-old to multi-million-year-old sediments?

What are their metabolic activities, and how do these activities affect their chemical and physical environment?

To address these questions effectively, a very wide range of sedimentological, geophysical, geochemical, and microbiological analyses were undertaken during Leg 201. To maximize understanding of the interplay between subsurface microorganisms and their environment, whenever possible, these diverse analyses were conducted on the same sediment samples or samples immediately adjacent to each other.

A full suite of standard sedimentary analyses was used to document the physical and compositional nature of subsurface environments explored during Leg 201. These included visual core descriptions, digital color scanning and optical reflectance scanning of the split cores, and microscopic observation and/or X-ray diffraction analyses of individual sediment samples.

Core logging of magnetic susceptibility and intensity was used to identify redox fronts of particular interest for Leg 201 objectives, such as pronounced biogeochemical fronts or lithostratigraphic boundaries. Core logs (magnetic susceptibility, gamma ray attenuation, and natural gamma radiation) were also used to correlate intervals of particular interest from hole to hole at the same site. Where possible, magnetic reversal logs were used to determine sediment age and correlate from hole to hole and site to site. Natural gamma radiation was measured both on cores in the laboratory and using wireline logs for in situ formation properties and was the physical property used to correlate between recovered and in situ sediment.

Analysis of the physical environment also included study of environmental properties such as temperature and pressure, which are important for the selection of cell properties and regulation of metabolic activity. Physical properties critical for quantifying transport processes, such as porosity and diffusivity, were analyzed in order to interpret the chemical gradients with respect to subsurface flow, chemical diffusion, and the availability of substrates for microorganisms.

The detailed analysis of pore water chemistry was a major emphasis during Leg 201 and was probably more comprehensive than during any previous ODP leg. A broad spectrum of dissolved inorganic ions, gases, and organic solutes was measured with close vertical resolution throughout the sediment column at each site in order to identify potential substrates and products of microbial metabolism and provide the chemical data necessary to quantitatively model steady-state net rates of microbial activities.

Additional shore-based analyses of solid-phase geochemistry and interstitial water chemistry will enable mass balance calculations of burial rates and diagenetic transformations of organic compounds and mineral phases. Stable isotope analyses of carbon, hydrogen, oxygen, sulfur, nitrogen, and iron in both dissolved and solid chemical phases will serve as a complementary approach to interpret the biogeochemical alterations at a time and depth scale that exceeds the detectability of modern experimental process studies. Furthermore, analyses of specific biomarkers and their isotopic signals will identify which organisms were involved in these slow alterations in the past.

A variety of experiments were undertaken during Leg 201 to estimate in situ activities of specific samples. Most of these experiments relied on minute quantities of radioactive chemicals to trace rates of specific activities. Although specific activities are generally limited to a subset of the microbial community, they typically serve a crucial role in the flow of energy and material through the entire community. Well-established 35S, 14C, and 3H techniques were used to quantify within-sample rates of sulfate reduction, methanogenesis, and thymidine uptake. Innovative experiments with 3H2 were used to trace H2 uptake and turnover. A few additional experiments included incubating samples with stable isotopes as tracers; some used 18O to study oxygen exchange between water and phosphate, and others used 13C to trace assimilation of carbon from acetate into biomass.

A wide variety of microbial cultivation experiments were initiated during Leg 201 to identify and quantify subseafloor microbial populations. These included a large number of incubations that utilize selective media for enrichments in order to isolate and identify a broad spectrum of physiological types with respect to energy metabolism and temperature adaptation (general heterotrophs; fermenters; autotrophic and heterotrophic sulfate reducers, methanogens, and acetogens; iron and manganese reducers; anaerobic ammonium and methane oxidizers; and psychrophiles, mesophiles, and thermophiles). Serial dilution (MPN) cultivation experiments were initiated to enumerate those organisms able to show growth and metabolic activity under nutrient-rich laboratory conditions. Homogenized sediment slurries were diluted in tenfold steps into liquid media, which should support the growth of specific physiological types of microorganisms. In such experiments, the highest dilutions that still have growth are classically interpreted to indicate the MPN of these organisms (American Public Health Association, 1989). MPN counts typically provide only a minimum estimate of the true numbers of organisms that were viable in situ because many microorganisms (perhaps the vast majority) are not cultivable with currently available methods. MPN cultivations also serve as starting material for enrichments and isolations of the organisms. The isolation of bacteria from the highest dilutions with positive growth maximizes the chance of finding organisms that are quantitatively dominant and, therefore, geochemically most important.

As for many ODP legs over the past decade, total cell numbers of microorganisms were determined during Leg 201 by direct microscopy of fluorescently stained cells (acridine orange direct count [AODC]). Similar counts of defined groups of organisms will be done by using fluorescence in situ hybridization (FISH) to specifically stain cells that share genetic sequence information.

The genetic diversity and geographic continuity of subseafloor microbial populations will be addressed by postcruise research. This research will largely rely on the recently developed approach of analyzing deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from entire communities in natural samples. This approach allows analysis of natural communities of microorganisms that are unavailable in culture, either because cultivation techniques have not yet succeeded in providing suitable growth conditions or because cultivation-based approaches have limited capacity to deal with great microbial diversity. DNA and RNA in Leg 201 sediment samples will be extracted by several participating groups, who will use them to analyze subseafloor microbial diversity based on genetic sequence information. This will for the first time establish a database on the diversity of microorganisms from the deep subsurface and the key genes of their energy metabolism.